Biomedical subjects

W M Casey

Publications and source records attributed to W M Casey.

13 recordsLinked to original sources

PubMed2001

Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification.

Large-scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost. In this regard, we have developed a rapid, cost-effective readout method for single nucleotide polymorphism (SNP) genotyping that combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs an ASPE reaction using PCR-derived target DNA containing the SNP and a pair of synthetic complementary capture probes that differ at their 3' end-nucleotide defining the alleles. Each capture probe has been synthesized to contain a unique 25-nucleotide identifying sequence (ZipCode) at its 5' end. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis. ASPE offers both an advantage of streamlining the SNP analysis protocol and an ability to perform multiplex SNP analysis on any mixture of allelic variants. All steps of the assay are simple additions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Comparison of the microsphere-based ASPE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.2% agreement in genotype assignments. In addition, the microsphere-based multiplex SNPs assay system was adapted for the identification of bacterial samples by both ASPE and single base chain extension (SBCE) assays. A series of probes designed for different variable sites of bacterial 16S rDNA permitted multiplex analysis and generated species- or genus-specific patterns. Seventeen bacterial species representing a broad range of gram-negative and gram-positive bacteria were analyzed within 16 variable sites of 16S rDNA sequence. The results were consistent with the published sequences and confirmed by direct DNA sequencing.

Alleles↗

PubMed1998

A novel bacterial reversion and forward mutation assay based on green fluorescent protein.

We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.

Aminacrine↗

PubMed1996

The activity of cytosolic phospholipase A2 is required for the lysis of adenovirus-infected cells by tumor necrosis factor.

Most cell types are resistant to apoptosis induced by tumor necrosis factor (TNF) unless the cells are treated with a sensitizing agent. Inhibitors of transcription or translation act as sensitizing agents, as do adenoviruses lacking one or more resistance genes. We have reported recently that the activity of cytosolic phospholipase A2 (cPLA2) is necessary for the TNF-induced lysis of cells that are sensitized by inhibitors of transcription or translation (C. Voelkel-Johnson, T. E. Thorne, and S. M. Laster, J. Immunol. 156:201-207, 1996). In this report we have asked whether the lysis of cells infected by the adenovirus dl758 (which lacks the E3 14.7-kDa resistance gene product) also involves the activity of cPLA2. We report that a phosphorothioate-modified antisense oligonucleotide specific for cPLA2, but not the control oligonucleotide, inhibited the TNF-induced release of both [3H]arachidonic acid and 51Cr from infected cells. Arachidonyltrifluoromethyl ketone (AA COCF3), an inhibitor of cPLA2, also inhibited the release of 51Cr, and we found that the release of [3H]arachidonic acid was highly selective and was preferred over the release of [3H]palmitic acid. Taken together, these results suggest strongly that cPLA2 is indeed the phospholipase responsible for the release of [3H]arachidonic acid during the lysis of infected cells and that its activity is necessary for cell death. Finally, since arachidonic acid serves as the substrate for the synthesis of inflammatory lipids, our results suggest a possible link between the TNF-induced lysis of infected cells and inflammation. The E3 14.7-kDa resistance protein may, therefore, play two roles: preventing TNF-induced cell death and, as our results show, preventing the TNF-induced release of arachidonic acid.

3T3 Cells↗

PubMed1995

Physiological implications of sterol biosynthesis in yeast.

Fungi are among the most primitive organisms that synthesize sterols. The fungal sterol, ergosterol, is similar to animal sterol, cholesterol, but with significant structural differences. The genetics and biochemistry for most of the steps in sterol biosynthesis have been studied in the yeast, Saccharomyces cerevisiae. Yet, little is known of the precise physiological roles that sterols play in the cell. Work with strains that are auxotrophic for ergosterol has led to the prediction of at least four growth-dependent functions for sterols. Most of the antifungal compounds in medical and agricultural use affect some aspect of sterol synthesis or function. Extensive studies on the modes of action of those substances and research on the effects of altering sterol metabolism by sterol mutants are providing new insights into sterol functions in the cells. In addition, questioning why fungi require ergosterol rather than the simpler cholesterol provides heuristic impetus for further experimentation.

Biological Transport↗

PubMed1994

Covalent attachment of palmitoleic acid (C16:1 delta 9) to proteins in Saccharomyces cerevisiae. Evidence for a third class of acylated proteins.

Saccharomyces cerevisiae was used as a model system to characterize the covalent attachment of palmitoleic acid to proteins. Chemically synthesized cis-[9,10-3H]hexadecenoic acid (palmitoleic acid) was used to demonstrate the attachment of this lipid species to at least six proteins (m = 122, 58, 45, 41, 31, and 17 kDa). The majority of the labeled proteins are distinct from those labeled with [3H]palmitic acid (16:0). Based on the lability of the bond in the presence of methanolic KOH or hydroxylamine (pH 8), we propose that [3H] palmitoleic acid is attached to proteins via a thioester linkage. The identity of the palmitoleic acid was established by C-18 reverse phase high performance thin layer chromatography and argentation thin layer chromatography analysis after the fatty acid was liberated from the proteins by either transesterification or saponification. Incorporation of [3H]palmitoleic acid into proteins was only slightly inhibited (relative to [3H] myristic acid) by the presence of cycloheximide, indicating that the attachment of [3H]palmitoleic acid occurs post-translationally. This report is the first description of multiprotein acylation by a long chain unsaturated fatty acid.

Acylation↗

PubMed1993

Effects of unsaturated fatty acid supplementation on phospholipid and triacylglycerol biosynthesis in Saccharomyces cerevisiae.

A fatty acid desaturase mutant was used to study the regulatory effects of unsaturated fatty acids on glycerolipid biosynthesis in yeast. Cells grown on palmitoleic acid (16:1) contain approximately twice the amount of phospholipids and triacylglycerols (per mg. dry weight) compared to those grown on oleic acid (18:1). The in vitro specific activity of glycerol-3-phosphate acyl transferase was two fold higher when palmitoyl-CoA was used as a substrate relative to oleoyl-CoA. In vivo methylation studies revealed that cells grown on palmitoleic acid produce 2.6 fold more phosphatidylcholine via the CDP-DAG (methylation) pathway than cells grown on oleic acid, although oleic acid facilitated the direct phosphorylation of exogenously supplied choline. These data indicate that unsaturated fatty acids may act as key regulatory molecules which influence the glycerolipid biosynthetic matrix in yeast.

Acetates↗

PubMed1992

Stimulation by heme of steryl ester synthase and aerobic sterol exclusion in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae sterol and heme auxotrophs were used to elucidate a role for hemes in sterol esterification. Steryl ester synthase (SES) activity was stimulated on average fourfold in cells supplemented with 50 micrograms/ml delta-aminolevulinic acid (ALA). This stimulation was not dependent on ALA per se, but on the ability of this precursor to effect heme competency. The addition of ALA stimulated SES activity of yeast on either fermentative or respiratory carbon sources. The elevation of SES activity was independent of intracellular free sterol, unsaturated fatty acid, or methionine levels. SES activity increases as the cells enter stationary phase, and this increase is enhanced by heme competency. SES was directly inhibited by the hypocholesterolemic drug lovastatin (mevinolin). The inhibition of SES activity by lovastatin was enhanced in heme-competent cells.

Acyltransferases↗

PubMed1992

Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.

5-Aminolevulinate Synthetase↗

PubMed1991

Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast.

We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described.

Genes, Fungal↗

PubMed1990

Saccharomyces cerevisiae membrane sterol modifications in response to growth in the presence of ethanol.

Membranes isolated from yeasts grown in the presence of ethanol do not display the thermally induced transition in diphenylhexatriene anisotropy that is seen in control cells when they are exposed to ethanol in vitro. The total sterol content of the cells that were exposed to ethanol during growth is reduced, with no steryl esters being detected. A greater proportion of the total sterol pool is ergosterol in cells grown in the presence of alcohol. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase is reduced by ethanol in vitro. Ethanol-exposed cells take up more exogenous sterol under aerobic conditions than do control cells. The presence of ethanol during growth reduces the activity of the plasma membrane enzyme, chitin synthase, as well as increasing the thermosensitivity of this enzyme.

Chitin Synthase↗

PubMed1989

A role for sterols in the porphyrin mediated photosensitization of yeast.

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.

Oxidation-Reduction↗

PubMed1989

Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.

Cholestanols↗

PubMedDate not supplied

Use of the green fluorescent protein to rapidly assess viability of E. coli in preserved solutions.

E. coli strain HB101 was genetically engineered to a fluorescent phenotype by transformation with a plasmid containing complementary DNA for a green fluorescent protein. The level of fluorescence in the transformed strain was directly proportional to the number of viable cells. There was a rapid decrease in fluorescence when transformed cells were inoculated into lamivudine solutions containing ten different preservative formulations. The decrease in fluorescence correlated to a decrease in the number of viable cells, allowing the relative antimicrobial properties of each solution to be compared. This methods provides a simple, rapid (< 2 min/assay), and accurate means of determining the effects of antimicrobial solutions on the viability of E. coli.

Drug Contamination↗